Jörg Kriegsmann

Anderson-Fabry disease: clinical manifestations of disease in female heterozygotes.

Anderson–Fabry disease is a rare, X-chromosomal lipid storage disorder caused by a deficiency of lysosomal α-galactosidase A. Clinical manifestations of Anderson–Fabry disease include excruciating pain in the extremities (acroparaesthesia), skin vessel ectasia (angiokeratoma), corneal and lenticular opacity, cardiovascular disease, stroke and renal failure, only renal failure being a frequent cause of death. Heterozygote female carriers have often been reported as being asymptomatic or having an attenuated form of the disease. To evaluate the spectrum of clinical signs in heterozygotes, a comprehensive clinical examination was performed on 20 carriers of Anderson–Fabry disease. This revealed that, in addition to the skin manifestation, various other clinical manifestations of the disease are present, including acroparaesthesia, kidney dysfunction, cerebrovascular disease, and gastrointestinal and heart problems. It therefore appears that Anderson–Fabry disease affects both hemizygotes and heterozyotes and therefore should be considered to be an X-linked dominant disease.

Proposal for a histopathological consensus classification of the periprosthetic interface membrane

Aims: The introduction of clearly defined histopathological criteria for a standardised evaluation of the periprosthetic membrane, which can appear in cases of total joint arthroplasty revision surgery. Methods: Based on histomorphological criteria, four types of periprosthetic membrane were defined: wear particle induced type (detection of foreign body particles; macrophages and multinucleated giant cells occupy at least 20% of the area; type I); infectious type (granulation tissue with neutrophilic granulocytes, plasma cells and few, if any, wear particles; type II); combined type (aspects of type I and type II occur simultaneously; type III); and indeterminate type (neither criteria for type I nor type II are fulfilled; type IV). The periprosthetic membranes of 370 patients (217 women, 153 men; mean age 67.6 years, mean period until revision surgery 7.4 years) were analysed according to the defined criteria. Results: Frequency of histopathological membrane types was: type I 54.3%, type II 19.7%, type III 5.4%, type IV 15.4%, and not assessable 5.1%. The mean period between primary arthroplasty and revision surgery was 10.1 years for type I, 3.2 years for type II, 4.5 years for type III and 5.4 years for type IV. The correlation between histopathological and microbiological diagnosis was high (89.7%), and the inter-observer reproducibility sufficient (85%). Conclusion: The classification proposed enables standardised typing of periprosthetic membranes and may serve as a tool for further research on the pathogenesis of the loosening of total joint replacement. The study highlights the importance of non-infectious, non-particle induced loosening of prosthetic devices in orthopaedic surgery (membrane type IV), which was observed in 15.4% of patients.

Biomaterial-induced sarcoma: A novel model to study preneoplastic change.

In the study of carcinogenesis most interest has focused on carcinomas, as they represent the majority of human cancers. The recognition of the adenoma-carcinoma sequence both in humans and in animal experimental models has given the field of basic oncology the opportunity to elucidate individual mechanisms in the multistep development of carcinoma. The relative scarcity of human sarcomas coupled with the lack of adequate animal models has hampered understanding of the molecular genetic steps involved. We present an experimental model in the rat in which a high incidence of malignant mesenchymal tumors arise around a subcutaneously implanted biomaterial. Nine commercially available biomaterials were implanted in a total of 490 rats of the Fischer strain for 2 years. On average, macroscopic tumors were found in 25.8% of implantation sites over a period from 26 to 110 weeks after implantation. The most frequent tumors were malignant fibrous histiocytomas and pleomorphic sarcomas, although fibrosarcomas, leiomyosarcomas, and angiosarcomas readily developed, the latter especially around polyurethane implants. Of particular interest are the results of a detailed histological study of the capsules around the implanted biomaterials without tumors. Here a spectrum of change from focal proliferative lesions through preneoplastic proliferation to incipient sarcoma could be observed. A parallel immunohistochemical study of peri-implant capsules showed that proliferating cell nuclear antigen was of particular help in identifying these atypical proliferative lesions. To our knowledge this is the first description of a sarcoma model in which preneoplastic lesions can be readily identified and also reproducibly induced. This model provides the molecular biologist with defined stages in the development of mesenchymal malignancy, with which the multistage tumorigenesis hypothesis can be tested, analogous to the well-known adenoma-carcinoma sequence.

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: Effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts

OBJECTIVE Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

CHARACTERIZATION OF COLLAGENASE 3 (MATRIX METALLOPROTEINASE 13) MESSENGER RNA EXPRESSION IN THE SYNOVIAL MEMBRANE AND SYNOVIAL FIBROBLASTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

OBJECTIVE To study the localization and cell type-specific expression of collagenase 3 messenger RNA (mRNA) in the synovial membrane, its regulation in primary synovial fibroblasts, and the correlation with systemic markers of inflammation and radiographic damage in rheumatoid arthritis (RA). METHODS The expression of collagenase 3 mRNA was characterized by Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization. Immunohistochemical detection of cell type-specific antigens was used in combination with in situ hybridization of collagenase 3 mRNA to characterize the cellular origin of collagenase 3 mRNA expression. RESULTS Collagenase 3 mRNA was detected in synovial membrane specimens of 21 of 36 RA patients (58%) and correlated with an increase in erythrocyte sedimentation rate (P<0.05) and C-reactive protein levels (P<0.005). Collagenase 3 mRNA was localized in fibroblast-like cells of the lining and sublining layers, and at the synovial membrane-cartilage interface. Four of 10 primary synovial fibroblast cell cultures showed basal expression of collagenase 3 mRNA, which was stimulated 2-4-fold upon interleukin-1beta or tumor necrosis factor alpha treatment and, in contrast to interstitial collagenase mRNA, 5-10-fold by increasing the intracellular level of cAMP. The stimulation by cAMP analogs was completely abolished by protein kinase A inhibitors. CONCLUSION Some RA patients show collagenase 3 mRNA expression in the synovial membrane, which correlates with elevated levels of systemic markers of inflammation in these patients. In synovial fibroblasts, the expression of collagenase 3 and interstitial collagenase mRNA is differentially regulated by distinct protein kinase signal transduction pathways.

MALDI TOF imaging mass spectrometry in clinical pathology: A valuable tool for cancer diagnostics (Review)

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) is an evolving technique in cancer diagnostics and combines the advantages of mass spectrometry (proteomics), detection of numerous molecules, and spatial resolution in histological tissue sections and cytological preparations. This method allows the detection of proteins, peptides, lipids, carbohydrates or glycoconjugates and small molecules.Formalin-fixed paraffin-embedded tissue can also be investigated by IMS, thus, this method seems to be an ideal tool for cancer diagnostics and biomarker discovery. It may add information to the identification of tumor margins and tumor heterogeneity. The technique allows tumor typing, especially identification of the tumor of origin in metastatic tissue, as well as grading and may provide prognostic information. IMS is a valuable method for the identification of biomarkers and can complement histology, immunohistology and molecular pathology in various fields of histopathological diagnostics, especially with regard to identification and grading of tumors.

Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis

ObjectiveThe aim of this study was to define and compare the expression of fibronectin (Fn) isoforms in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA).MethodsUsing monoclonal antibodies specific for total Fn, extra domain (ED)-A Fn, ED-B Fn, and oncofetal glycosylated Fn, we studied the expression of the Fn isoforms in synovium. Furthermore, in situ hybridization for the detection of ED-B Fn mRNA including a double labeling technique for the detection of cell type was applied.ResultsStrong expression of total Fn, ED-A Fn, oncofetal glycosylated Fn and, to a lesser extent, ED-B Fn could be demonstrated in the synovial lining layer in both RA and OA. Stromal and vessel expression of Fn isoforms was more prominent in RA tissue. Pannus tissue showed strong labeling with ED-B Fn.ConclusionThe expression of alternatively spliced isoforms of Fn is associated with tissue remodeling and, as a partial process of this phenomenon, with neovascularization rather than underlying disease, X-ray status, or parameters of acute inflammation. In the lining layer, Fn expression correlates with hyperplasia associated with cell recruitment but not with proliferative status. Most remarkably, the expression of ED-B Fn in pannus tissue seems to be associated with the invasive phenotype described in RA tissue.

New aspects in the histological examination of polyethylene wear particles in failed total joint replacements

The most important long-term complication in total joint replacements is aseptic osteolysis. Wear particles such as polyethylene (PE) debris are considered to be one of the causes that play a central role. Several studies indicated that PE can be visualised in paraffin-embedded tissue sections not only by polarised light, but also after oil red staining. To determine whether oil red staining enables sensitive detection of PE, we examined staining of mechanically-produced PE particles by oil red. Furthermore, we studied oil red staining of paraffin-embedded tissue specimens of patients with failed uncemented and cemented total knee and hip prostheses. We applied double labelling of sections by immunohistochemistry using the macrophage marker anti-CD68 and oil red staining. We found that oil red stains both isolated PE particles and PE particles in paraffin-embedded tissue sections. Polymethylmethacrylate particles in failed cemented arthroplasties did not stain in paraffin sections. Double labelling showed strong colocalisation of CD68 and PE. We suggest that oil red staining is a sensitive method to detect PE particles. Oil red staining is particularly helpful in these cases which show a characteristic histological feature of aseptic prosthesis loosening without particles being detectable with routine microscopy and polarised light. We also established that immunohistochemical methods can be applied together with the oil red staining method.

Imaging mass spectrometry to discriminate breast from pancreatic cancer metastasis in formalin-fixed paraffin-embedded tissues.

Diagnosis of the origin of metastasis is mandatory for adequate therapy. In the past, classification of tumors was based on histology (morphological expression of a complex protein pattern), while supportive immunohistochemical investigation relied only on few “tumor specific” proteins. At present, histopathological diagnosis is based on clinical information, morphology, immunohistochemistry, and may include molecular methods. This process is complex, expensive, requires an experienced pathologist and may be time consuming. Currently, proteomic methods have been introduced in various clinical disciplines. MALDI imaging MS combines detection of numerous proteins with morphological features, and seems to be the ideal tool for objective and fast histopathological tumor classification. To study a special tumor type and to identify predictive patterns that could discriminate metastatic breast from pancreatic carcinoma MALDI imaging MS was applied to multitissue paraffin blocks. A statistical classification model was created using a training set of primary carcinoma biopsies. This model was validated on two testing sets of different breast and pancreatic carcinoma specimens. We could discern breast from pancreatic primary tumors with an overall accuracy of 83.38%, a sensitivity of 85.95% and a specificity of 76.96%. Furthermore, breast and pancreatic liver metastases were tested and classified correctly.

Susceptibility to collagen-induced arthritis is modulated by TGFβ responsiveness of T cells

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor β (TGFβ) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFβ type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice than in wild-type mice (57% vs 35%, P < 0.05). Affected transgenic animals displayed a significantly higher clinical (4.5 ± 0.6 vs 1.67 ± 0.19, P = 0.001) and histological arthritis score (8.01 ± 0.9 vs 4.06 ± 1.1, P < 0.05). Draining lymph node cells of transgenic mice secreted more tumor necrosis factor α and IFNγ and proliferated more vigorously in response to collagen type II and upon CD3/CD28 costimulation in vitro. Therefore, the regulation of T cells by endogenous TGFβ is important for the maintenance of joint integrity after arthritis induction. Defects in TGFβ-signalling as a susceptibility factor for rheumatoid arthritis may warrant further investigation.